The present invention relates to an improved method of stabilizing polyacrylamide gels of various types. More specifically, this invention relates to a method for stabilizing rehydratable polyacrylamide gels with substituted monosaccharides or oligosaccharides. These rehydratable polyacrylamide gels can be stored for extended periods of time at ambient temperatures, and can be rehydrated without loss of structural or functional integrity to be used for electrophoretic separation of proteins and nucleic acids.
Polyacrylamide gels are used as a support matrix for the electrophoretic separation of proteins, double-stranded DNA, and single stranded RNA. Typically, polyacrylamide gels are prepared individually just prior to use as homogeneous or gradient pore size gels. Precast wet gels bonded to polyester sheets can be obtained commercially. These precast polyacrylamide gels must be stored in sealed containers to prevent evaporation.
Rehydratable polyacrylamide gels bonded to a polyester support have been disclosed in the art (see, for example, U.S. Pat. No. 4,746,551, issued to Allen, et al., 1988). The patent teaches that the rehydratable polyacrylamide gels can be stabilized with polyols, polymeric alcohols, polyamines or high molecular weight polysaccharides such as dextran. The protective action of these stabilizers appears to occur on the surface of the polyacrylamide gels. These stabilizers are too large to penetrate the matrix and pores of the polyacrylamide gel to any great degree, i.e., not more than about 3% of the polysaccharide stabilizes the gel to control residual wetness.
It has been found to be desirable to store these gels in containers, otherwise the pore spaces may collapse from loss of residual moisture with resultant shrinking of the gel when the relative humidity falls below 70%. This can cause curling of the polyester backing and eventual peeling of the gel from the backing. Also, as humidity rises, the surface of the gels can hydrate, and the gels can become stuck to the packing material with surface destruction when the gels are peeled away for use. Thus, storage can be a problem, and care must be taken when storing the rehydratable gels.
Other methods at attempting to prepare and/or stabilize gels for electrophoretic use can be found in U.S. Pat. Nos. 3,875,044, 4,006,069, and 4,189,370. U.S. Pat. No. 3,875,044, issued to Renn et al., discloses hydratable gel sheets and methods for preparing them. The gel sheets can be prepared from agar, agarose or gelling carrageenan. The gel is joined to a support backing by drying the gel sheet to the support backing such that a bond is formed between the two components so that the gel sheet does not slide from the support backing. Thus, a relatively firm and rigid structure can be formed which maintains its shape and dimensions. The hydratable gels then can be used in immunodiffusion, continuous zone electrophoresis (CZE), or chromatographic procedures. The gels disclosed in this patent are limited to CZE as opposed to other forms of electrophoresis because there is considerable electroendosmotic flow from the materials which comprise the gels, i.e., agar, agarose and carrageenan. Also, because the pore sizes of the gels described in this patent are relatively large, separation of small DNA and RNA is limited, and many macromolecules will migrate with the boundaries, thus preventing multizonal electrophoresis (MZE).
U.S. Pat. No. 4,006,069, issued to Hiratsuka et al., discloses an electrophoretic support made of a cellulosic material such as cellulose acetate. The pores of the support are infiltrated with a polymeric gel membrane composed of polyacrylamide or starch or agar. The polymeric gel membrane is tough and reportedly does not break during use or substantially shrink upon drying after electrophoresis (CZE). However, cellulose acetate has charged pores which cause electroendosmotic flow; thus, isoelectric focusing is impossible with these gels. The cellulose acetate also prevents silver staining for proteins and nucleic acids. Further, the large pore sizes of these gels prevents the separation of small DNA and RNA as well as MZE electrophoresis.
U.S. Pat. No. 4,189,370, issued to Boschetti, discloses a process for preparing plates made of a gel-polymer for the electrophoretic separation of serif or plasmatic lipoproteins. The gel-polymer can be made by radical polymerization of N-methylol-acrylamide and a bifunctional allylic or acrylic cross-linking agent. The plates described herein can be treated with a mixture of a simple sugar, a diol or a triol and a carboxylic polysaccharide, previously treated by a reducing agent, may be air-dried, then rehydrated to regain its original characteristics. These gels have several disadvantages. Because the gel is made with a substituted polyacrylamide which contains a free charge group, electroendosmotic flow characteristics are imparted to the gel, thereby making it unsuitable for the separation of macromolecules by isoelectric focusing. In addition, the low molecular weight polyols he discloses, such as glycerol and ethylene glycol, can intersect with nucleic acids and proteins, thereby altering their mobility in moving boundary systems. They thus are unsuitable as preservatives, as discussed in more detail below.
Attempts have been made to improve the stabilization of rehydratable gels using polyhydroxy alcohols, or linear sugar alcohols typically employed as matrix modifiers in gels employed in the separation of double-stranded DNA. Allen, et al., "Rehydratable Gels: A Potential Reference Standard Support For Electrophoresing PCR-Amplified DNA" Biotechnology, 8:1288 (1990), and Allen, et al., "Tailoring DNA Resolution in Polyacrylamide Gels with Electrochemistry and Pulsed Constant Power" Electrophoresis ,'89, page 91 (1989). These compounds can be used to alter the mobility and resolution of nucleic acids in a given pore size gel. This characteristic renders them unsuitable for the stabilization of an inert or empty gel. These matrix modifiers, in particular glycerol, tend to make the gel surface sticky and will glue to the protective film covering the gels during storage. Frey, et al., "Preparation of Rehydratable Polyacrylamide Gels and their Application in Ultrathin-Layer Isoelectric Focusing" Electrochoresis 1986, vol. 7, pp. 28-40 (1986). Preferably, glycerol is removed from rehydratable gels by washing with distilled water prior to drying the gels for storage. Gelfi, et al., "Swelling Kinetics of Immobiline Gels for Isoelectric Focusing" Electrophoresis, vol 5, pp. 257-262 (1984).
In addition to glycerol, the sugar alcohols used as matrix modifiers, such as sorbitol and mannitol, also have been found unsuitable as preservatives. Polyols, such as ethylene glycol and glycerol and the sugar alcohols, such as sorbitol and mannitol, interact with nucleic acids, altering mobility in high resolution moving boundary electrophoresis. In addition, glycerol recently has been shown to interact not only with nucleic acids, but also with proteins and enzymes. For example, the mobility of erythrocyte acid phosphatase is altered in a manner such that two of the isotypes or isoforms are no longer distinguishable in its presence. Allen, et al., "Enzyme and Antibody Detection Following Isoelectric Focusing on Ultrathin-Layer Rehydrated Polyacrylamide Gels" Acta Histochem. Cytochem., Vol. 19, No. 5, pp. 637-645 (1986).
Therefore, there exists a need for a method of stabilizing rehydratable polyacrylamide gels such that the integrity of the gel is maintained for electrophoretic separation of proteins, enzymes, DNA, RNA and the like throughout storage at ambient temperatures and upon rehydration prior to use, and the stabilizers employed do not significantly interact with the substrates to be separated.